Recombinant Production of Heparin Binding Proteins

ABSTRACT

A process for recovering and purifying refolded heparin binding proteins produced in heterologous host cells includes the step of incubation of the solubilized protein with a polyanionic species such as dextran sulfate.

RELATED APPLICATION

This application claims priority to and the benefit of U.S. ProvisionalApplication Ser. No. 60/753,615, filed Dec. 22, 2005, and U.S.Provisional Application Ser. No. 60/807,432, filed Jul. 14, 2006, thespecifications of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to methods for obtaining heparin-binding proteinsproduced in cell culture. The invention includes methods for recoveringand purifying refolded heparin binding proteins that have been producedin prokaryotic host cells and are present in these cells, typically inthe periplasmic or intracellular space. The heparin binding proteinsproduced in prokaryotic host cells can also be found as soluble proteinsor a mixture of soluble and insoluble proteins.

BACKGROUND

It is known that a large variety of naturally occurring, biologicallyactive polypeptides bind heparin. Such heparin-binding polypeptidesinclude cytokines, such as platelet factor 4 and IL-8 (Barber et al.,(1972) Biochim. Biophys. Acta, 286:312-329; Handin et al., (1976) J.Biol. Chem., 251:4273-422; Loscalzo et al., (1985) Arch. Biochem.Biophys. 240:446-455; Zucker et al., (1989) Proc. Natl. Acad. Sci. USA,86:7571-7574; Talpas et al., (1991) Biochim. Biophys. Acta,1078:208-218; Webb et al., (1993) Proc. Natl. Acad. Sci. USA,90:7158-7162) heparin-binding growth factors (Burgess and Maciag, (1989)Annu. Rev. Biochem., 58:576-606; Klagsbrun, (1989) Prog. Growth FactorRes., 1:207-235), such as epidermal growth factor (EGF);platelet-derived growth factor (PDGF); basic fibroblast growth factor(bFGF); acidic fibroblast growth factor (aFGF); vascular endothelialgrowth factor (VEGF); and hepatocyte growth factor (HGF) (Liu et al.,(1992) Gastrointest. Liver Physiol. 26:G642-G649); and selecting, suchas L-selectin, E-selectin and P-selectin (Norgard-Sumnicht et al.,(1993) Science, 261:480-483). See also, Munoz and Linhardt., (2004)Arterioscler Thromb Vasc Biol., 24:1549-1557.

International Publication No. WO 95/07097 describes formulations ofheparin binding proteins including heparin binding growth factors suchas VEGF, with purified native heparin or other polyanionic compounds fortherapeutic use. Heparin derived oligosaccharides and various otherpolyanionic compounds have been shown to stabilize the activeconformation for heparin binding growth factors (Barzu et al., (1989) J.Cell. Physiol. 140:538-548; Dabora et al., (1991) J. Biol. Chem.266:23627-23640) and heparin affinity chromatography has been employedin various purification schemes (see generally, InternationalPublication No. WO 96/02562).

Many of the heparin binding proteins of mammalian origin have beenproduced by recombinant technology and are clinically relevant (Munozand Linhardt, (2004) Arterioscler Thromb Vasc Biol., 24:1549-1557;Favard et al. (1996) Diabetes and Metabolism 22(4):268-73; Matsuda etal., (1995) J. Biochem. 118(3):643-9; Roberts et al., (1995) BrainResearch 699(1):51-61). For example, VEGF is a potent mitogen forvascular endothelial cells. It is also known as vascular permeabilityfactor (VPF). See, Dvorak et al., (1995) Am. J. Pathol. 146:1029-39.VEGF play important roles in both vasculogenesis, the development of theembryonic vasculature, and angiogenesis, the process of forming newblood vessels from pre-existing ones. See, e.g., Ferrara, (2004)Endocrine Reviews 25(4):581-611; Risau et al., (1988) Dev. Biol.,125:441-450; Zachary, (1998) Intl. J. Biochem Cell Bio 30:1169-1174;Neufeld et al., (1999) FASEB J. 13:9-22; Ferrara (1999) J. Mol. Med.77:527-543; and, Ferrara and Davis-Smyth, (1997) Endocri. Rev. 18:4-25.Clinical applications for VEGF include those where the growth of newcapillary beds is indicated as, for example, in promoting wound healing(see, for example, International Publication No. WO 91/02058; and,Attorney Docket No. P2358R1, entitled “Wound Healing” filed on Jun. 16,2006), in promoting tissue growth and repair, e.g., liver (see, e.g.,WO2003/0103581), bone (see, e.g., WO2003/094617), etc. See also,Ferrara, (2004) Endocrine Reviews 25(4):581-611.

Typically, therapeutically relevant recombinant proteins are produced ina variety of host organisms. Most proteins can be expressed in theirnative form in eukaryotic hosts such as CHO cells. Animal cell culturegenerally requires prolonged growing times to achieve maximum celldensity and ultimately achieves lower cell density than prokaryotic cellcultures (Cleland, J. (1993) ACS Symposium Series 526, Protein Folding:In Vivo and In Vitro, American Chemical Society). Additionally, animalcell cultures often require expensive media containing growth componentsthat may interfere with the recovery of the desired protein. Bacterialhost expression systems provide a cost-effective alternative to themanufacturing scale production of recombinant proteins. Numerous U.S.patents on general bacterial expression of recombinant proteins exist,including U.S. Pat. Nos. 4,565,785; 4,673,641; 4,795,706; and 4,710,473.A major advantage of the production method is the ability to easilyisolate the product from the cellular components by centrifugation ormicrofiltration. See, e.g., Kipriyanov and Little, (1999) MolecularBiotechnology, 12: 173-201; and, Skerra and Pluckthun, (1988) Science,240: 1038-1040.

Recombinant heparin binding growth factors such as acidic fibroblastgrowth factor, basic fibroblast growth factor and vascular endothelialgrowth factor have been recovered and purified from a number of sourcesincluding bacteria (Salter D. H. et al., (1996) Labor. Invest.74(2):546-556 (VEGF); Siemeister et al., (1996) Biochem. Biophys. Res.Commun. 222(2):249-55 (VEGF); Cao et al., (1996) J. Biol. Chem.261(6):3154-62 (VEGF); Yang et al., (1994) Gaojishu Tongxun, 4:28-31(VEGF); Anspach et al., (1995) J. Chromatogr. A 711(1):129-139 (aFGF andbFGF); Gaulandris (1994) J. Cell. Physiol. 161(1):149-59 (bFGF); Estapeand Rinas (1996) Biotech. Tech. 10(7):481-484 (bFGF); McDonald et al.,(1995) FASEB J. 9(3):A410 (bFGF)). However, bacterial expression systemssuch as E. coli lack the cellular machinery to facilitate properrefolding of the proteins and generally do not result in the secretionof large proteins into the culture media. Recombinant proteins expressedin bacterial host cells are often found as inclusion bodies consistingof dense masses of partially folded and misfolded reduced protein. Inthis form, the recombinant protein is generally inactive. For example,the predominant active form of VEGF is a homodimer of two 165-amino acidpolypeptides (VEGF-165). In this structure, each subunit contains 7pairs of intrachain disulfide bonds and two additional pairs whicheffect the covalent linkage of the two subunits (Ferrara et al., (1991)J. Cell. Biochem. 47:211-218). The native conformation includes astrongly basic domain which has been shown to readily bind heparin(Ferrara et al (1991) supra). Covalent dimerization of VEGF is neededfor effective receptor binding and biological activity (Potgens et al.,(1994) J. Biol. Chem. 269:32879-32885; Claffey et al., (1995) Biochim.et Biophys. Acta 1246:1-9). The bacterial product potentially containsseveral misfolded and disulfide scrambled intermediates.

Additionally, refolding often produces misfolded and disulfide-linkeddimers, trimers, and multimers. (Morris et al., (1990) Biochem. J.,268:803-806; Toren et al., (1988) Anal. Biochem., 169:287-299). Thisassociation phenomenon is very common during protein refolding,particularly at higher protein concentrations, and appears often toinvolve association through hydrophobic interaction of partially foldedintermediates (Cleland and Wang, (1990) Biochemistry, 29:11072-11078).

Misfolding occurs either in the cell during fermentation or during theisolation procedure. Proteins recovered from periplasmic orintracellular space must be solubilized and the soluble protein refoldedinto the native state. In vitro methods for refolding the proteins intothe correct, biologically active conformation are essential forobtaining functional proteins. Typical downstream processing of proteinsrecovered from inclusion bodies includes the dissolution of theinclusion body at high concentration of a denaturant such as ureafollowed by dilution of the denaturant to permit refolding to occur(see, U.S. Pat. Nos. 4,512,922; 4,511,502; and 4,511,503). See also,e.g., Rudolph and Lilie, (1996) FASEB J. 10:49-56; and, Fischer et al.,(1993), Biotechnology and Bioengineering, 41:3-13. Such recovery methodsare regarded as being universally applicable, with minor modifications,to the recovery of biologically active recombinant proteins frominclusion bodies. These methods have been applied to heparin bindingprotein such as VEGF (Siemeister et al. (1996) supra). These methodsseek to eliminate random disulfide bonding prior to coaxing therecombinant protein into its biologically active conformation throughits other stabilizing forces and may not eliminate improperly foldedintermediates or provide homogenous populations of properly foldedproduct.

Reversed micelles or ion exchange chromatography have been used toassist refolding of denatured proteins by enclosing a single proteinwithin micelles or isolating them on a resin and then removing thedenaturant (Hagen et al., (1990) Biotechnol. Bioeng. 35:966-975;Creighton (1985) in Protein Structure Folding and Design (Oxender, D. L.Ed.) pp. 249-251, New York: Alan R. Liss, Inc.). These methods have beenuseful in preventing protein aggregation and facilitating properrefolding. To alter the rate or extent of refolding,conformation-specific refolding has been performed with ligands andantibodies to the native structure of the protein (Cleland and Wang,(1993), in Biotechnology, (Rehm H.-J., and Reed G. Eds.) pp 528-555, NewYork, VCH). For example, creatine kinase was refolded in the presence ofantibodies to the native structure (Morris et al., (1987) Biochem. J.248:53-57). In addition to antibodies, ligands and cofactors have beenused to enhance refolding. These molecules would be more likely tointeract with the folding protein after formation of the native protein.Therefore, the folding equilibrium could be “driven” to the nativestate. For example, the rate of refolding of ferricytochrome c wasenhanced by the extrinsic ligand for the axial position of the heme iron(Brems and Stellwagon, (1983) J. Biol. Chem. 258:3655-3661). Chaperoneproteins have also been used to assist with protein folding. See, e.g.,Baneyx, (1999) Current Opinion in Biotechnology, 10:411-421.

There is a need for new and more effective methods of folding and/orrecovering heparin binding proteins from a host cell culture, e.g., forthe efficient and economical production of heparin binding proteins inbacterial cell culture that provides for elimination or reduction ofbiologically inactive intermediates and improved recovery of a highlypurified biologically active properly refolded protein and that isgenerally applicable to manufacturing scale production of the proteins.The invention addresses these and other needs, as will be apparent uponreview of the following disclosure.

SUMMARY OF THE INVENTION

The invention provides a method for recovering and purifying refoldedheparin binding proteins from cell culture. In particular the inventionprovides a method of recovering a heparin binding protein fromprokaryotic host cells, e.g., bacterial cells. For example, a methodcomprises the steps of (a) isolating insoluble heparin binding proteinfrom the periplasmic or intracellular space of said bacterial cells; (b)solubilizing said isolated insoluble heparin binding protein in a firstbuffered solution comprising a chaotropic agent and a reducing agent,and c) incubating said solubilized heparin binding protein in a secondbuffered solution comprising a chaotropic agent and a sulfatedpolyanionic agent for such a time and under such conditions thatrefolding of the heparin binding protein occurs; and (d) recovering saidrefolded heparin binding protein, wherein there is a 2 to 10 foldincrease in protein concentration recovered by incubating with asulfated polyanionic agent compared to a control. In one embodiment, thesecond buffered solution further comprises arginine. In one embodiment,the second buffered solution further comprises cysteine or a mildreducing agent.

In one embodiment of the invention, there is a, e.g., 2-8 fold increasein protein concentration of recovered biologically active refoldedprotein, or 2-5 fold increase in protein concentration of recoveredbiologically active refolded protein, or 3-5 fold increase in proteinconcentration of recovered biologically active refolded protein, or a2-3 fold increase in protein concentration of recovered biologicallyactive refolded protein. In another embodiment of the invention, thereis a, e.g., greater than a 2.0 fold, a 2.5 fold, a 2.8 fold, a 3.0 fold,a 5-fold, a 6 fold, a 7.0 fold, an 8 fold, a 9 fold, etc., increase inprotein concentration recovered of biologically active refolded protein.In one embodiment of the invention, there is a 3 to 5-fold increase inprotein concentration of biologically active refolded VEGF.

The processes of the invention are broadly applicable to heparin bindingproteins and especially to heparin binding growth factors and inparticular, vascular endothelial growth factor (VEGF). In certainembodiments of the invention, the sulfated polyanionic agent is betweenabout 3,000 and 10,000 daltons. In one embodiment, the sulfatedpolyanionic agent utilized in the production processes is a dextransulfate, sodium sulfate or heparin sulfate. In one aspect, the dextransulfate is between 3,000 daltons and 10,000 daltons.

The invention additionally provides processes and methods forpurification of heparin binding proteins either alone or in connectionwith the recovery of the heparin binding protein as described herein. Ina particular embodiment, purification methods include contacting saidrefolded heparin binding protein with a hydroxyapatite chromatographicsupport; a first hydrophobic interaction chromatographic support, acationic chromatographic support and a second hydrophobic interactionchromatographic support and selectively eluting the heparin bindingprotein from each support. In another embodiment, a purification methodcomprises contacting said refolded heparin binding protein with a cationexchange support; a first hydrophobic interaction chromatographicsupport, and an ion exchange or mixed-media chromatographic support andselectively eluting the heparin binding protein from each support. It iscontemplated that the steps for recovery steps can be performed in anyorder, e.g., sequentially or altering the order of the chromatographicsupports. In certain embodiments of the invention, methods are providedfor recovering and purifying refolded heparin binding proteins frommanufacturing or industrial scale cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a chromatograph from VEGF produced by bacterialstrain W3110 loaded on a POROS HE2/M column (4.6×100 mm, PerSeptiveBioResearch Products, Cambridge, Mass.). For example, the POROS HE/2Mcolumn is equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 Msodium chloride. The column is eluted using a linear gradient from0.15-2 M sodium chloride in, 10 mM sodium phosphate, pH 7 over 10minutes. The eluant is monitored at 280 nm. The protein recovered ineach peak corresponds to VEGF however only peak 3 corresponds to abiologically active properly refolded VEGF.

FIG. 2 illustrates a graph depicting the stabilization of nativeproperly folded VEGF by heparin. The VEGF is suspended in 50 mM HEPES,pH 8, containing 5 mM EDTA, 0.2 M NaCl and 10 mM cysteine.

FIGS. 3A-3D illustrates chromatographs from VEGF produced by bacterialstrain W3110 and incubated with 12 μg/ml dextran sulfate 5,000 daltons(FIG. 3A); 12 μg/ml dextran sulfate 8,000 daltons (FIG. 3B); 12 μg/mldextran sulfate 10,000 daltons (FIG. 3C) or 25 μg/ml heparin (FIG. 3D),3,000 daltons and loaded on a POROS HE2/M column (4.6×100 mm, PerSeptiveBioResearch Products, Cambridge, Mass.). For example, the column isequilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M sodiumchloride. The column is eluted using a linear gradient from 0.15-2 Msodium chloride in, 10 mM sodium phosphate, pH 7 over 10 minutes. Theeluant is monitored at 280 nm. The protein recovered in each peakcorresponds to VEGF however only peak 3 corresponds to a biologicallyactive properly refolded VEGF.

FIG. 4 illustrates the effect of scale on the refolding of VEGF.

FIG. 5 illustrates the effect of heparin, low molecular weight (MW) andhigh MW, and dextran sulfate, 10,000 daltons, on VEGF refolding. Peak 3corresponds to a biologically active properly refolded VEGF.

FIG. 6 illustrates the effect of sodium sulfate on VEGF refolding. Peak3 corresponds to a biologically active properly refolded VEGF.

FIG. 7 illustrates the effect of heparin, low molecular weight (MW) andhigh MW, and dextran sulfate, 5,000 daltons, 8,000 daltons, and 10,000daltons, on VEGF refolding. Peak 3 corresponds to a biologically activeproperly refolded VEGF.

FIG. 8 illustrates the effect of heparin and dextran sulfate on VEGFrefolding. Peak 3 corresponds to a biologically active properly refoldedVEGF.

FIG. 9 illustrates an effect of urea and DTT on the extraction of VEGFfrom bacterial inclusion bodies.

FIG. 10 illustrates an effect of urea and DTT concentration on therefolding of VEGF.

FIG. 11 illustrates the amino acid sequence of VEGF₁₆₅ with disulfidebonds indicated (SEQ ID NO.: 1).

FIG. 12 illustrates the effect of the presence of charged amino acids.At 0.75M concentration in the second buffered solution both arginine andlysine are beneficial whereas histidine has little additive effect ascompared to the buffered solution without it. Additionally arginine hasbeen shown to have similar effect at concentrations of 0.1 to 1M.

FIG. 13 illustrates the effect of dilution in the % refold efficiency,where, although the total VEGF concentration is lower as the dilutionincreases, the % refold efficiency is higher with more dilution.

DETAILED DESCRIPTION Definitions

“Heparin” (also referred to as heparinic acid) is a heterogenous groupof highly sulfated, straight-chain anionic mucopolysaccharides, calledglycosaminoglycans. Although others may be present, the main sugars inheparin are: α-L-iduronic acid 2-sulfate, 2-deoxy-2-sulfamino-α-glucose6-sulfate, β-D-glucuronic acid, 2-acetamido-2-deoxy-α-D-glucose, andL-iduronic acid. These and optionally other sugars are joined byglycosidic linkages, forming polymers of varying sizes. Due to thepresence of its covalently linked sulfate and carboxylic acid groups,heparin is strongly acidic. The molecular weight of heparin varies fromabout 3,000 to about 20,000 daltons depending on the source and themethod of determination.

Native heparin is a constituent of various tissues, especially liver andlung, and mast cells in several mammalian species. Heparin and heparinsalts (heparin sodium) are commercially available and are primarily usedas anticoagulants in various clinical situations.

“Dextran sulfate” is a sulfate of dextran whose principal structure is apolymer of D-glucose. Glucose and optionally other sugars are joined byα-D(1-6) glycosidic linkages, forming polymers of varying sizes. Due tothe presence of covalently linked sulfate, dextran sulfate is stronglyacidic. The sulfur content is generally not less than 10%, and typicallyabout 15%-20% with up to 3 sulfate groups per glucose molecule. Theaverage molecular weight of dextran sulfate is from about 1,000 to about40,000,000 daltons. Examples of dextran sulfate employable in theinvention include the sulfate of the dextrans produced frommicroorganisms such as Leuconostoc mesenteroides and L. dextranicum.

“Polyanionic agent” as used within the scope of the invention is meantto describe commercially available purified native heparin preparationsand compounds which are capable of binding to heparin binding proteinsincluding other “polyanionic agents” such as sodium sulfate, heparinsulfate, heparan sulfate, pentosan (poly) sulfate, dextran, dextransulfate, hyaluronic acid, chondroitin, chondroitin sulfate, dermatansulfate, and keratan sulfate. Particularly useful within the context ofthe invention is a “sulfated polyanionic agent,” such as for example, asulfate derivative of a polysaccharide, such as heparin sulfate, dextransulfate, the sulfates of the cyclodextrin produced by microorganismssuch as Bacillus macerans described in U.S. Pat. No. 5,314,872 as wellas sulfates of other glucans such as β-1,3 glucan sulfates, the β-1,3glucan being produced by microorgansims belonging to the genusAlcaligenes or Agrobacterium, and chondroitin sulfate as well assulfated heparin fragments.

The above mentioned agents are generally available and recognized by theskilled artisan. For example, sulfated heparin fragments may be obtainedfrom a library of heparin-derived oligosaccharides that have beenfractionated by gel-permeation chromatography. The preparation ofaffinity-fractionated, heparin-derived oligosaccharides was reported byIshihara et al., (1993) J. Biol. Chem., 268:4675-4683. Theseoligosaccharides were prepared from commercial porcine heparin followingpartial depolymerization with nitrous acid, reduction with sodiumborohydride, and fractionation by gel permeation chromatography. Theresulting pools of di-, tetra-, hexa-, octa-, and decasaccharides weresequentially applied to an affinity column of human recombinant bFGFcovalently attached to SEPHAROSE™ 4B, and were further fractionated intosubpools based on their elution from this column in response togradients of sodium chloride. This resulted in five pools, designatedHexa-1 to Hexa-5, the structures and biological activities of which werefurther evaluated. The structure of Hexa-5C and its 500-MHz NMR spectrumare shown in FIG. 4 of Tyrell et al., (1993) J. Biol. Chem.,268:4684-4689. This hexasaccharide has the structure[IdoA(2-OSO₃)α1-4GlcNSO₃(6-OSO₃)α1-4]₂IdoA(2-OSO₃)α1-4AMan_(R)(6-OSO₃).All heparin-derived oligosaccharides discussed above, as well as otherheparin-like oligosaccharides are suitable for and can be used inaccordance with the invention. In one embodiment of the invention,hexasaccharides and polysaccharides of heparin of higher unit size (e.g.hepta-, octa-, nona- and decasaccharides) are used. Furthermore,heparin-derived or heparin-like oligosaccharides with a large netnegative charge, e.g. due to a high degree of sulfation, are used withadvantage.

The term “heparin-binding protein” or “HPB” as used herein refers to apolypeptide capable of binding heparin (as hereinabove defined). Thedefinition includes the mature, pre, pre-pro, and pro forms of nativeand recombinantly produced heparin-binding proteins. Typical examples ofheparin-binding proteins are “heparin binding growth factors,” includingbut not limited to epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF) (also known as scatter factor,SF), and nerve growth factor (NGF), IL-8, etc.

As used herein, “vascular endothelial growth factor”, or “VEGF”, refersto a mammalian growth factor derived originally from bovine pituitaryfollicular cells having the amino acid sequence disclosed in Castor, C.W., et al., (1991) Methods in Enzymol. 198:391-405, together withfunctional derivatives thereof having the qualitative biologicalactivity of a corresponding native VEGF, including, but not limited to,the human VEGF amino acid sequence as reported in Houck et al., (1991)Mol. Endocrin. 5:1806-1814. See also, Leung et al. (1989) Science,246:1306, and, Robinson & Stringer, (2001) Journal of Cell Science,144(5):853-865, U.S. Pat. No. 5,332,671. The predominant form of VEGF isa 165 amino acid homodimer having sixteen cysteine residues that form 7intramolecular disulfide bonds and two intermolecular disulfide bonds.Alternative splicing has been implicated in the formation of multiplehuman VEGF polypeptides consisting of 121, 145, 165, 189 and 206 aminoacids, however the VEGF₁₂₁ variant lacks the heparin binding domain ofthe other variants and therefore does not fall within the definition ofheparin binding protein set forth herein. All isoforms of VEGF share acommon amino-terminal domain, but differ in the length of thecarboxyl-terminal portion of the molecule. The preferred active form ofVEGF, VEGF₁₆₅, has disulfide bonds between amino acid residuesCys26-Cys68; Cys57-Cys104; Cys61-Cys102; Cys117-Cys135; Cys120-Cys137;Cys139-Cys; 158; Cys146-Cys160 in each monomer. See FIG. 11. See also,e.g., Keck et al., (1997) Archives of Biochemistry and Biophysics344(1): 103-113. The VEGF₁₆₅ molecule is composed of two domains: anamino-terminal receptor-binding domain (amino acids 1-110 disulfidelinked homodimer) and a carboxyl-terminal heparin-binding domain(residues 111-165). See, e.g., Keyt et al., (1996) J. Biol. Chem.,271(13):7788-7795. In certain embodiments of the invention, the VEGF₁₆₅isolated and purified is not glycosylated at residue 75 (Asn). See,e.g., Yang et al., (1998) Journal of Pharm. & Experimental Therapeutics,284:103-110. In certain embodiments of the invention, the VEGF₁₆₅isolated and purified is substantially undeamidated at residue Asn10. Incertain embodiments of the invention, the VEGF₁₆₅ isolated and purifiedis a mixture of deamidated (at residue Asn10) and undeamidated protein,typically with majority of the protein being undeamidated. Since VEGF₁₆₅is a homodimer, deamination can occur on one or both polypeptide chains.

As used herein “properly folded” or “biologically active” VEGF or otherHBP and the like refers to a molecule with a biologically activeconformation. The skilled artisan will recognize that misfolded anddisulfide scrambled intermediates may have biological activity. In sucha case the properly folded or biologically active VEGF or HBPcorresponds to the native folding pattern of the VEGF (described above)or other HBP. For example, properly folded VEGF has the above noteddisulfide pairs, in addition to two intermolecular disulfide bonds inthe dimeric molecule however other intermediates may be produced bybacterial cell culture (FIGS. 1 and 3A-3D). For properly folded VEGF thetwo intermolecular disulfide bonds occur between the same residues,Cys51 and Cys60, of each monomer. See, e.g., WO98/16551 patent.Biological activities of VEGF include, but are not limited to, e.g.,promoting vascular permeability, promoting growth of vascularendothelial cells, binding to a VEGF receptor, binding and signalingthrough a VEGF receptor (see, e.g., Keyt et al., (1996) Journal ofBiological Chemistry, 271(10):5638-5646), inducing angiogenesis, etc.

The terms “purified” or “pure HBP” and the like refer to a material freefrom substances which normally accompany it as found in its recombinantproduction and especially in prokaryotic or bacterial cell culture. Thusthe terms refer to a recombinant HBP which is free of contaminantingDNA, host cell proteins or other molecules associated with its in situenvironment. The terms refer to a degree of purity that is at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95% or at least about 98% or more.

The terms “inclusion bodies” or “refractile bodies” refer to denseintracellular masses of aggregated polypeptide of interest, whichconstitute a significant portion of the total cell protein, includingall cell components. In some cases, but not all cases, these aggregatesof polypeptide may be recognized as bright spots visible within theenclosure of the cells under a phase-contrast microscope atmagnifications down to 1,000 fold.

As used herein, the term “misfolded” protein refers to precipitated oraggregated polypeptides that are contained within refractile bodies. Asused herein, “insoluble” or “misfolded” VEGF or other HBP refers toprecipitated or aggregated VEGF that is contained within the periplasmor intracellular space of prokaryotic host cells, or is otherwiseprokaryotic host cell associated, and assumes a biologically inactiveconformation with mismatched or unformed disulfide bonds. The insolubleHBP is generally, but need not be, contained in refractile bodies, i.e.,it may or may not be visible under a phase contrast microscope.

As used herein, “chaotropic agent” refers to a compound that, in asuitable concentration in aqueous solution, is capable of changing thespatial configuration or conformation of polypeptides throughalterations at the surface thereof so as to render the polypeptidesoluble in the aqueous medium. The alterations may occur by changing,e.g., the state of hydration, the solvent environment, or thesolvent-surface interaction. The concentration of chaotropic agent willdirectly affect its strength and effectiveness. A strongly denaturingchaotropic solution contains a chaotropic agent in large concentrationswhich, in solution, will effectively unfold a polypeptide present in thesolution effectively eliminating the proteins secondary structure. Theunfolding will be relatively extensive, but reversible. A moderatelydenaturing chaotropic solution contains a chaotropic agent which, insufficient concentrations in solution, permits partial folding of apolypeptide from whatever contorted conformation the polypeptide hasassumed through intermediates soluble in the solution, into the spatialconformation in which it finds itself when operating in its active formunder endogenous or homologous physiological conditions. Examples ofchaotropic agents include guanidine hydrochloride, urea, and hydroxidessuch as sodium or potassium hydroxide. Chaotropic agents include acombination of these reagents, such as a mixture of a hydroxide withurea or guanidine hydrochloride.

As used herein, “reducing agent” refers to a compound that, in asuitable concentration in aqueous solution, maintains free sulfhydrylgroups so that the intra- or intermolecular disulfide bonds arechemically disrupted. Representative examples of suitable reducingagents include dithiothreitol (DTT), dithioerythritol (DTE),beta-mercaptoethanol (BME), cysteine, cysteamine, thioglycolate,glutathione, Tris[2-carboxyethyl]phosphine (TCEP), and sodiumborohydride.

As used herein, “buffered solution” refers to a solution which resistschanges in pH by the action of its acid-base conjugate components.

The “bacteria” for purposes herein include eubacteria andarchaebacteria. In certain embodiments of the invention, eubacteria,including gram-positive and gram-negative bacteria, are used in themethods and processes described herein. In one embodiment of theinvention, gram-negative bacteria are used, e.g., Enterobacteriaceae.Examples of bacteria belonging to Enterobacteriaceae includeEscherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella,Serratia, and Shigella. Other types of suitable bacteria includeAzotobacter, Pseudomonas, Rhizobia, Vitreoscilla, and Paracoccus. In oneembodiment of the invention, E. coli is used. Suitable E. coli hostsinclude E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coliB. and E. coli X1776 (ATCC 31,537). These examples are illustrativerather than limiting, and W3110 is one example. Mutant cells of any ofthe above-mentioned bacteria may also be employed. It is, of course,necessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well-known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. See further below regardingexamples of suitable bacterial host cells.

As used herein, the expressions “cell,” “cell line,” “strain,” and “cellculture” are used interchangeably and all such designations includeprogeny. Thus, the words “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polypeptide” refers generally to peptides and proteinsfrom any cell source having more than about ten amino acids.“Heterologous” polypeptides are those polypeptides foreign to the hostcell being utilized, such as a human protein produced by E. coli. Whilethe heterologous polypeptide may be prokaryotic or eukaryotic,preferably it is eukaryotic, more preferably mammalian, and mostpreferably human. In certain embodiments of the invention, it is arecombinantly produced, or recombinant polypeptide.

Heparin Binding Proteins

Isolating Heparin Binding Protein

Insoluble, misfolded heparin binding protein (HBP) is isolated fromprokaryotic host cells expressing the protein by any of a number of artstandard techniques. For example, the insoluble HBP is isolated in asuitable isolation buffer by exposing the cells to a buffer of suitableionic strength to solubilize most host proteins, but in which thesubject protein is substantially insoluble, or disrupting the cells soas to release the inclusion bodies or the protein form the periplasmicor intracellular space and make them available for recovery by, forexample, centrifugation. This technique is well known and is describedin, for example, U.S. Pat. No. 4,511,503. Kleid et al., disclosepurification of refractile bodies by homogenization followed bycentrifugation (Kleid et al., (1984) in Developments in IndustrialMicrobiology, (Society for Industrial Microbiology, Arlington, Va.)25:217-235). See also, e.g., Fischer et al., (1993) Biotechnology andBioengineering 41:3-13.

U.S. Pat. No. 5,410,026 describes a typical method for recoveringprotein from inclusion bodies and is summarized as follows. Theprokaryotic cells are suspended in a suitable buffer. Typically thebuffer consists of a buffering agent suitable for buffering at betweenpH 5 to 9, or about 6 to 8 and a salt. Any suitable salt, includingNaCl, is useful to maintain a sufficient ionic strength in the bufferedsolution. Typically an ionic strength of about 0.01 to 2 M, or 0.1 to0.2 M is employed. The cells, while suspended in this buffer, aredisrupted or lysed using techniques commonly employed such as, forexample, mechanical methods, e.g., Homogenizer (Manton-Gaulin press,Microfluidizer, or Niro-Soavi), a French press, a bead mill, or a sonicoscillator, or by chemical or enzymatic methods.

Examples of chemical or enzymatic methods of cell disruption includespheroplasting, which entails the use of lysozyme to lyse the bacterialwall (H. Neu et al., (1964) Biochem. Biophys. Res. Comm., 17:215), andosmotic shock, which involves treatment of viable cells with a solutionof high tonicity and with a cold-water wash of low tonicity to releasethe polypeptides (H. Neu et al., 1965 J. Biol. Chem., 240(9):3685-3692).Sonication is generally used for disruption of bacteria contained inanalytical scale volumes of fermentation broth. At larger scales highpressure homogenization is typically used.

After the cells are disrupted, the suspension is typically centrifugedat low speed, generally around 500 to 15,000×g, e.g., in one embodimentof the invention about 12,000×g is used, in a standard centrifuge for atime sufficient to pellet substantially all of the insoluble protein.Such times can be simply determined and depend on the volume beingcentrifuged as well as the centrifuge design. Typically about 10 minutesto 0.5 hours is sufficient to pellet the insoluble protein. In oneembodiment the suspension is centrifuged at 12,000×g for 10 minutes.

The resulting pellet contains substantially all of the insoluble proteinfraction. If the cell disruption process is not complete, the pellet mayalso contain intact cells or broken cell fragments. Completeness of celldisruption can be assayed by resuspending the pellet in a small amountof the same buffer solution and examining the suspension with a phasecontrast microscope. The presence of broken cell fragments or wholecells indicates that further sonication or other means of disruption isnecessary to remove the fragments or cells and the associatednon-refractile polypeptides. After such further disruption, if required,the suspension is again centrifuged and the pellet recovered,resuspended, and reexamined. The process is repeated until visualexamination reveals the absence of broken cell fragments in the pelletedmaterial or until further treatment fails to reduce the size of theresulting pellet.

The above process can be employed whether the insoluble protein isintracellular or in the periplasmic space. In one embodiment of theinvention, the conditions given herein for isolating heparin bindingprotein are directed to inclusion bodies precipitated in the periplasmicspace or intracellular space and relate particularly to VEGF. However,the processes and procedures are thought to be applicable to heparinbinding proteins in general with minor modifications as noted throughoutthe following text. In certain embodiments of the invention, theprocesses and procedures are applicable to manufacturing or industrialscale production, refolding, and purification of the HBP.

Refolding Heparin Binding Proteins

The isolated insoluble, misfolded heparin binding protein is incubatedin a first buffered solution containing an amount of a chaotropic agentand a reducing agent sufficient to substantially solubilize the heparinbinding protein. This incubation takes place under conditions ofconcentration, incubation time, and incubation temperature that willallow solubilization of some or substantially all the heparin bindingprotein, and for unfolding to occur.

Measurement of the degree of solubilization in the buffered solution canbe simply determined and is suitably carried out, for example, byturbidity determination, by analyzing fractionation between thesupernatant and pellet after centrifugation, on reduced SDS-PAGE gels,by protein assay (e.g., the Bradford reagent protein assay (e.g.,Pierce, Bio-Rad etc.)), or by HPLC.

The first buffered solution comprises a buffering agent suitable formaintaining the pH range of the buffer at least about 7.0, with thetypical range being 7.5-10.5. In one embodiment, the pH for VEGF is pH8.0. Examples of suitable buffers that will provide a pH within thislatter range include TRIS-HCl (Tris[hydroxymethyl]aminomethane),HEPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propane-sulfonic acid]), HEPES(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP(2-Amino-2-methyl-1-propanol), CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES(2-[N-Cyclohexylamino]ethanesulfonic acid), glycine, and sodium acetate.In one embodiment of the invention, the buffer herein is HEPPS at aboutpH 8.0. In a further embodiment, the buffers, e.g., such as HEPPS, aresulfated.

Chaotropic agents suitable for practicing this invention include, e.g.,urea and salts of guanidine or thiocyanate, e.g., urea, guanidinehydrochloride, sodium thiocyanate, etc. The amount of chaotropic agentnecessary to be present in the buffer is an amount sufficient to unfoldthe HBP in solution. In certain embodiments of the invention, achaotrope is present at about between about 4 and 10 molar. In oneembodiment of the invention, the chaotropic agent is urea at about 5-8M, or at about 7 M. In another example, the chaotropic agent isguanidine hydrochloride at about 6-8 M.

Examples of suitable reducing agents include, but are not limited to,dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethonol (BME),cysteine, DTE, etc. The amount of reducing agent to be present in thebuffer will depend mainly on the type of reducing agent and chaotropicagent, the type and pH of the buffer employed, the amount of oxygenentrained in or introduced to the solution, and the concentration of theprotein in the buffer. For example, with 0.5-1.5 mg/ml protein in abuffered solution at pH 7.0-10.0 containing 4-8 M urea, and reducingagent is, e.g., DTT with a concentration at about 1-15 mM, or BME with aconcentration at about 0.2-2 mM, or cysteine with a concentration atabout 2-10 mM. In one embodiment, the reducing agent is DTT at about 0.5to about 4 mM, or 2-4 mM. FIG. 9 illustrates the effect of urea and DTTon the extraction of VEGF. Peak 3 VEGF refers to properly foldedbiologically active VEGF. In one embodiment, the reducing agent is DTTat about 10 mM. A single reducing agent or a combination of reducingagents can be used in a buffer herein.

The concentration of the protein in the buffered solution must be suchthat the protein will be substantially solubilized as determined byoptical density. The exact amount to employ will depend on, e.g., theconcentrations and types of other ingredients in the buffered solution,particularly the protein concentration, reducing agent, and the pH ofthe buffer. In one embodiment of the invention, the concentration ofheparin binding protein is in the range of 0.5-5.5 mg per ml, or 1.5-5.0mg/ml. The solubilization is typically carried out at about 0-45° C., orabout 20-40° C., or about 23-37° C., or about 25-37° C., or about 25° C.for at least about one to 24 hours. In one embodiment, thesolubilization is carried out for at least about two hours at roomtemperature. Typically, the temperature is not apparently affected bysalt, reducing agent and chaotropic agent levels.

After the polypeptide is solubilized, it is placed or diluted into asecond buffered solution containing the chaotropic agent and a sulfatedpolyanionic agent as described above however at a concentration ofchaotropic agent which allows for refolding of the heparin bindingprotein.

The conditions of this second incubation of the soluble, misfoldedprotein will generally be such that some or substantial or completerefolding of the protein will take place. The exact conditions willdepend on, for example, the pH of the buffer and the types andconcentrations of sulfated polyanionic agents and of chaotropic andreducing agents, if any, present. The incubation temperature isgenerally about 0-40° C., or 10-40° C. and the incubation will generallybe carried out for at least about 1 hour to effect refolding. In certainembodiments, the reaction is carried out, e.g., at about 15-37° C., orat 20-30° C., for at least about 6 hours, for at least about 10 hours,or between about 10 and 48 hours, or between about 15 and 20 hours, orbetween 6 and 20 hours, or between 12 and 24 hours.

The degree of refolding is suitably determined by radio-immuno assay(RIA) titer of the HPB or by high performance liquid chromatography(HPLC) analysis using e.g., a POROS HE2/M column (PerSeptive BioResearchProducts) or other appropriate heparin affinity column. Increasing RIAtiter or correctly folded HBP peak size directly correlates withincreasing amounts of correctly folded, biologically active HPB presentin the buffer. The incubation is carried out to maximize the ratio ofcorrectly folded HPB to misfolded HPB recovered, as determined by RIA orHPLC.

In one embodiment, the quality and quantity of properly-folded VEGF isassessed using a heparin-binding assay. Samples containing the dilutedheparin binding protein are loaded on a e.g., POROS HE2/M column(4.6×100 mm, PerSeptive BioResearch Products, Cambridge, Mass.) or othersuitable heparin affinity column. For example, the heparin affinitycolumn is equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 Msodium chloride. At a flow rate of 1 ml/min or 2 ml/min, the column iseluted using a linear gradient from 0.15-2 M sodium chloride in, 10 mMsodium phosphate, pH 7 over 10 minutes. The eluant is monitored at 280nm. In one embodiment, the protein is recovered in a single peakcorresponding to the biologically active properly refolded HBP. In oneembodiment of the invention, an assay for determining properly refoldedHBP is RPHPLC. Disulfide linkages can optionally be confirmed by peptidemap. Circular dichroism can also be used in for determining 2 & 3Dstructure/folding.

The buffer for the second buffered solution can be any of those listedabove for the first buffered solution, e.g., HEPPS pH. 8.0, e.g., at aconcentration of about 50 mM for refolding VEGF. The polypeptide may bediluted with the refolding buffer, e.g., at least five fold, or at leastabout ten fold, about 20 fold, or about 40 fold. Alternatively, thepolypeptide may be dialyzed against the refolding buffer.

The second buffered solution contains a chaotropic agent at aconcentration such that refolding of the HPB occurs. Generally achaotrope is present at about between about 0.5 and 2 molar. In oneembodiment of the invention, the chaotropic agent herein is urea atabout 0.5-2 M, 0.5-2 M, or at about 1 M. In one embodiment, thechaotropic agent is urea at about 1.3 M concentration. In anotherembodiment of the invention, the chaotropic agent is guanidinehydrochloride at about 1 M. FIG. 10 illustrates the effect of urea andreducing agent DTT on the refolding of VEGF. Peak 3 VEGF refers toproperly folded biologically active VEGF.

As noted, the solution optionally also contains a reducing agent. Thereducing agent is suitably selected from those described above for thesolubilizing step in the concentration range of about 0.5 to about 10 mMfor cysteine, 0.1-1.0 mM for DTT, and/or less than about 0.2 mM for BME.In one embodiment of the invention, the reducing agent is DTT at about0.5-2 mM. In one embodiment of the invention, the reducing agent is DTTat about 0.5 mM. Examples of suitable reducing agents include, but arenot limited to, e.g., dithiothreitol (DTT), β-mercaptoethonol (BME),cysteine, DTE, etc. Whereas DTT and BME can be used in connection withthe procedures provided herein for heparin binding proteins in general,a combination of cysteine at about 0.1 to about 10 mM and about 0.1 toabout 1.0 mM DTT as described herein is an example for the recovery ofVEGF.

The refolding step includes a sulfated polyanionic agent at aconcentration sufficient to achieve complete refolding of thesolubilized protein. Examples of suitable polyanionic agents aredescribed herein above, e.g., a sulfate derivative of a polysaccharideas noted above with sulfated polyanionic agents such as heparin sulfate,dextran sulfate, heparin sulfate, and chrondroitin sulfate as well assulfated heparin fragments. For heparin sulfates used in the context ofthe invention, the molecular weight are generally between about 3,000and 10,000 daltons, or between about 3,000 and 6,000 dalton.

In one embodiment of the invention, dextran sulfate is employed in thecontext of the invention. The molecular weight of the sulfatedpolyanionic or other agent such as dextran sulfate employed in theinvention depends upon the size of the particular heparin bindingprotein being recovered. Generally, dextran sulfate between about 3,000and 10,000 daltons is employed. In one embodiment of the invention,dextran sulfate between about 5,000 daltons and 10,000 daltons is used,e.g., for the recovery of VEGF. In another embodiment, a dextran sulfatebetween about 5,000 and 8,000 daltons is used for recovery of the HBP.FIG. 3A-3D shows the recovery of VEGF with various concentrations of andmolecular weights of dextran sulfate (FIGS. 3A-C) and heparin (FIG. 3D)as analyzed by heparin affinity chromatography. Peak 3 corresponds toproperly folded VEGF.

The concentration of the polyanionic compound employed depends upon theprotein being recovered and its concentration and conditions such astemperature and pH of the refolding buffer. Typical concentrations arebetween about 50 and 500 mM for sodium sulfate, between about 10 and 200μg/ml for low molecular weight heparins such as 6,000 dalton heparin(Sigma Chemical Co.), between about 10 and 200 μg/ml for high molecularweight heparins such as porcine heparin I-A (Sigma Chemical Co.) andbetween about 10 and 400 μg/ml, or between about 10 and 200 μg/ml fordextran sulfates.

The refolding buffer can optionally contain additional agents such asany of a variety of non-ionic detergents such as TRITON™ X-100, NONIDET™P-40, the TWEEN™ series and the BRIJ™ series. The non-ionic detergent ispresent at about between 0.01% and 1.0%. In one example, theconcentrations for non ionic detergent are between about 0.025% and0.05%, or about 0.05%.

Optionally, positively charged amino acids, e.g., arginine (e.g.,L-arginine/HCl), lysine, etc., can be present in the refolding buffer.In certain embodiments of the invention, the concentration of arginineis e.g., about 0-1000 mM, or about 25 to 750 mM, or about 50-500 mM, orabout 50-250 mM, or about 100 mM final concentration, etc. In certainembodiments of the invention, the protein is in a buffer solution at pH7.0-9.0 containing, 0.5-3 M urea, 0-30 mg/L dextran sulfate, 0-0.2%Triton X-100, 2-15 mM cysteine, 0.1-1 mM DTT and 0-750 mM arginine,final concentration. In one embodiment, 50 mM HEPPS is used. In oneembodiment, the final concentration of the refolding buffer solution is1 M urea, 50 mM HEPPS, 15 mg/L dextran sulfate, 0.05% Triton X-100, 7.5mM cysteine, 100 mM arginine, pH 8.0. In one embodiment, the finalconcentration of the refolding buffer solution is 1.3 M urea, 50 mMHEPPS, 15 mg/L dextran sulfate, 0.05% Triton X-100, 7.5 mM cysteine, 0.5mM DTT, 100 mM arginine, pH 8.0.

Recovery and Purification of Heparin Binding Proteins

Although recovery and purification of the heparin binding protein fromthe culture media can employ various methods and known procedures forthe separation of such proteins such as, for example, salt and solventfractionation, adsorption with colloidal materials, gel filtration, ionexchange chromatography, affinity chromatography, immunoaffinitychromatography, electrophoresis and high performance liquidchromatography (HPLC), an example of a four step chromatographicprocedure is described which comprises contacting said refolded heparinbinding protein with a hydroxyapatite chromatographic support; a firsthydrophobic interaction chromatographic support, a cationicchromatographic support and a second hydrophobic interactionchromatographic support and selectively eluting the heparin bindingprotein from each support. Alternatively, another chromatographicprocedure is described which comprises contacting said refolded heparinbinding protein with a cation exchange support; a hydrophobicinteraction chromatographic support, and an ion exchange chromatographicsupport and selectively eluting the heparin binding protein from eachsupport. It is contemplated that the steps of either procedure can beperformed in any order. In one embodiment of the invention, the stepsare performed sequentially.

A suitable first step in the further recovery and purification of theheparin binding protein characteristically provides for theconcentration of the heparin binding protein and a reduction in samplevolume. For example, the second incubation step described above, mayresult in a large increase in the volume of the recovered heparinbinding protein and concornitant dilution of the protein in therefolding buffer. Suitable first chromatographic supports provide areduction in volume of recovered heparin binding protein and mayadvantageously provide some purification of the protein from unwantedcontaminating proteins. Suitable first chromatographic steps includechromatographic supports which can be eluted and loaded directly onto afirst hydrophobic interaction chromatographic support. For example,chromatographic supports from which the heparin binding protein can beeluted in a high salt concentration suitable for loading a hydrophobicinteraction chromatographic support are used.

Exemplary first chromatographic supports include, but are not limitedto, hydroxyapatite chromatographic supports, e.g., CHT ceramic type Iand type II (formally known as MacroPrep ceramic), Bio-Gel HT, Bio-GelHTP, Biorad, Hercules, Calif., etc.; metal chelating chromatographicsupports consisting of an inert resin of immobilized metal ions such ascopper, nickel, etc.; as well as non-derivatized silica gels. In oneembodiment of the invention, the first chromatographic supports for thepurification and recovery of VEGF are hydroxyapatite chromatographicsupports. In another embodiment of the invention, the firstchromatographic supports for the purification and recovery of VEGF arecation exchange supports, e.g., described below in more detail.

Elution from the first chromatographic support is accomplished accordingto art standard practices. Suitable elution conditions and buffers willfacilitate the loading of the eluted HPB directly onto the firsthydrophobic interaction chromatographic support as described below.

Hydrophobic interaction chromatography is well known in the art and ispredicated on the interaction of hydrophobic portions of the moleculeinteracting with hydrophobic ligands attached to “chromatographicsupports.” A hydrophobic ligand coupled to a matrix is variouslyreferred to as an HIC chromatographic support, HIC gel, or HIC columnand the like. It is further appreciated that the strength of theinteraction between the protein and the HIC column is not only afunction of the proportion of non-polar to polar surfaces on the proteinbut of the distribution of the non-polar surfaces as well.

A number of matrices may be employed in the preparation of HIC columns.The most extensively used is agarose, although silica and organicpolymer resins may be used. Useful hydrophobic ligands include but arenot limited to alkyl groups having from about 2 to about 10 carbonatoms, such as butyl, propyl, or octyl, or aryl groups such as phenyl.Conventional HIC supports for gels and columns may be obtainedcommercially from suppliers such as GE Healthcare, Uppsala, Sweden underthe product names butyl-SEPHAROSE™, phenyl-SEPHAROSE™ CL-4B, octylSEPHAROSE™ FF and phenyl SEPHAROSE™ FF and Tosoh Corporation, Tokyo,Japan under the product names TOYOPEARL™ butyl 650M (Fractogel TSKButyl-650) or TSK-GEL phenyl 5PW. In one embodiment, the purificationand recovery of VEGF is a first HIC chromatographic support that isbutyl-agarose and a second hydrophobic chromatographic support that is aphenyl agarose. In another embodiment, the first HIC chromatographicsupport is phenyl agarose.

Ligand density is an important parameter in that it influences not onlythe strength of the interaction of the protein but the capacity of thecolumn as well. The ligand density of the commercially available phenylor octyl phenyl gels is on the order of 5-40 μmol/ml gel bed. Gelcapacity is a function of the particular protein in question as well aspH, temperature and salt concentration but generally can be expected tofall in the range of 3-20 mg/ml gel.

The choice of particular gel can be determined by the skilled artisan.In general the strength of the interaction of the protein and the HICligand increases with the chain length of the alkyl ligands but ligandshaving from about 4 to about 8 carbon atoms are suitable for mostseparations. A phenyl group has about the same hydrophobicity as apentyl group, although the selectivity can be different owing to thepossibility of pi-pi interaction with aromatic groups of the protein.

Adsorption of the protein to a HIC column is favored by high saltconcentration, but the actual concentration can vary over a wide rangedepending of the nature of the protein and the particular HIC ligandchosen. In general salt concentration between about 1 and 4 M areuseful.

Elution from an HIC support, whether stepwise or in the form of agradient, can be accomplished in a variety of ways such as a) bychanging the salt concentration, b) by changing the polarity of thesolvent or c) by adding detergents. By decreasing salt concentrationsadsorbed proteins are eluted in order of increasing hydrophobicity.Changes in polarity may be effected by additions of solvents such asethylene glycol or isopropanol thereby decreasing the strength of thehydrophobic interactions. Detergents function as displacers of proteinsand have been used primarily in connection with the purification ofmembrane proteins.

Various anionic constituents may be attached to matrices in order toform cationic supports for chromatography. Anionic constituents includecarboxymethyl, sulfethyl groups, sulfopropyl groups, phosphate andsulfonate (S). Cellulosic ion exchange resins such as SE52 SE53, SE92,CM32, CM52, CM92, P11, DE23, DE32, DE52, EXPRESS ION™ S and EXPRESS ION™C are available from Whatman LTD, Maidstone Kent U.K. SEPHADEX™ andSEPHAROSE™ based and cross linked ion exchangers are also known underthe product names CM SEPHADEX™ C-25, CM SEPHADEX™ C-50 and SP SEPHADEX™.C-25 SP SEPHADEX™ C-50 and SP-SEPHAROSE™ High Performance, SP-SEPHAROSE™Fast Flow, SP-SEPHAROSE XL, CM-SEPHAROSE™ Fast Flow, and CM-SEPHAROSE™,CL-6B, all available from GE Healthcare. Examples of ion exchangers forthe practice of the invention include but are not limited to, e.g., ionexchangers under the product names MACROPREP™ such as for exampleMACROPREP™ S support, MACROPREP™ High S support and MACROPREP™ CMsupport from BioRad, Hercules, Calif.

Elution from cationic chromatographic supports is generally accomplishedby increasing salt concentrations. Because the elution from ioniccolumns involves addition of salt and because, as mentioned, HIC isenhanced in salt concentration the introduction of HIC step followingthe ionic step or other salt step is optionally used. In one embodimentof the invention, a cationic exchange chromatographic step precede theHIC step.

Examples of methods for purifying VEGF is described herein below, e.g.,see Example V and VI. After refolding, insoluble material in the pool isremoved by depth filtration. The clarified pool is then loaded on to aceramic hydroxyapatite (Bio Rad, Hercules, Calif.) equilibrated in 5-mMHEPPS/0.05% TRITON™ X100/pH 8. The non-binding protein is removed bywashing with equilibration buffer and the VEGF eluted using an isocraticstep of 50 mM HEPPS/0.05% TRRITON™ X100/0.15 M sodium phosphate/pH 8.The pool of VEGF is loaded onto a column of Butyl SEPHAROSE™ Fast Flow(GE Healthcare, Uppsala, Sweden) equilibrated in 50 mM HEPPS/0.05%TRITON™ X100/0.15 M sodium phosphate/pH 8. The column is washed withequilibration buffer and the VEGF collected in the column effluent. TheButyl SEPHAROSE™ pool is loaded onto a column of Macro Prep High S(BioRad, Hercules, Calif.) that is equilibrated in 50 mM HEPES/pH 8.After washing the effluent absorbance at 280 nm to baseline, the columnis washed with two column volumes of 50 mM HEPES/0.25 M sodiumchloride/pH 8. The VEGF is eluted using a linear, 8-column-volumegradient from 0.25-0.75 M sodium chloride in 50 mM HEPES/pH 8. Fractionsare collected and those which contained properly-folded VEGF, asdetermined by a heparin-binding assay, are pooled.

The Macro Prep High S pool is conditioned with an equal volume of 50 mMHEPES/0.8 M sodium citrate/pH 7.5. The conditioned pool is then loadedon to a column of Phenyl 5PW TSK (Tosoh Bioscience LLC, Montgomeryville,Pa.) that is equilibrated with 50 mM HEPES/0.4 M sodium citrate/pH 7.5.After washing non-binding protein through the column with equilibrationbuffer, the VEGF is eluted from the column using a 10-column-volumegradient from 0.4-0 M sodium citrate in 50 mM HEPES, pH 7.5. Fractionsare assayed by SDS-polyacrylamide gel electrophoresis and thosecontaining VEGF of sufficient purity pooled.

Expressing Heparin Binding Protein in Host Cells

In brief, expression vectors capable of autonomous replication andprotein expression relative to the host prokaryotic cell genome areintroduced into the host cell. Construction of appropriate expressionvectors is well known in the art including the nucleotide sequences ofthe heparin binding proteins described herein. See, e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y.) (2001); Ausubel et al.,Short Protocols in Molecular Biology, Current Protocols John Wiley andSons (New Jersey) (2002); and, Baneyx, (1999) Current Opinion inBiotechnology, 10:411-421. Appropriate prokaryotic cell, includingbacteria, expression vectors are available commercially through, forexample, the American Type Culture Collection (ATCC), Rockville, Md.Methods for the large scale growth of prokaryotic cells, and especiallybacterial cell culture are well known in the art and these methods canbe used in the context of the invention.

For example, prokaryotic host cells are transfected with expression orcloning vectors encoding the heparin binding protein of interest andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The nucleic acid encoding thepolypeptide of interest is suitably RNA, cDNA, or genomic DNA from anysource, provided it encodes the polypeptide(s) of interest. Methods arewell known for selecting the appropriate nucleic acid for expression ofheterologous polypeptides (including variants thereof) in microbialhosts. Nucleic acid molecules encoding the polypeptide are prepared by avariety of methods known in the art. For example, a DNA encoding VEGF isisolated and sequenced, e.g., by using oligonucleotide probes that arecapable of binding specifically to the gene encoding VEGF.

The heterologous nucleic acid (e.g., cDNA or genomic DNA) is suitablyinserted into a replicable vector for expression in the microorganismunder the control of a suitable promoter. Many vectors are available forthis purpose, and selection of the appropriate vector will depend mainlyon the size of the nucleic acid to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components depending on the particular host cell withwhich it is compatible. Depending on the particular type of host, thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, a promoter, and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with microbial hosts. The vector ordinarily carries areplication site, as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (see, e.g., Bolivar et al., (1977) Gene, 2: 95). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other bacterial plasmid or phage, also generally contains,or is modified to contain, promoters that can be used by the host forexpression of the selectable marker genes.

(i) Signal Sequence

Polypeptides of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is typically a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected typically isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native polypeptide signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders.

(ii) Origin of Replication Component

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of microbes. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria such asE. coli.

(iii) Selection Gene Component

Expression vectors generally contain a selection gene, also termed aselectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Thisselectable marker is separate from the genetic markers as utilized anddefined by this invention. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies other than those caused by the presence of the geneticmarker(s), or (c) supply critical nutrients not available from complexmedia, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. In this case, those cells that are successfully transformedwith the nucleic acid of interest produce a polypeptide conferring drugresistance and thus survive the selection regimen. Examples of suchdominant selection use the drugs neomycin (Southern et al., (1982) J.Molec. Appl. Genet. 1: 327), mycophenolic acid (Mulligan et al., (1980)Science 209: 1422) or hygromycin (Sugden et al., (1985) Mol. Cell.Biol., 5: 410-413). The three examples given above employ bacterialgenes under eukaryotic control to convey resistance to the appropriatedrug G418 or neomycin (geneticin), xgpt (mycophenolic acid), orhygromycin, respectively.

(iv) Promoter Component

The expression vector for producing the heparin binding protein ofinterest contains a suitable promoter that is recognized by the hostorganism and is operably linked to the nucleic acid encoding thepolypeptide of interest. Promoters suitable for use with prokaryotichosts include the beta-lactamase and lactose promoter systems (Chang etal., (1978) Nature, 275: 615; Goeddel et al., (1979) Nature, 281: 544),the arabinose promoter system (Guzman et al., (1992) J. Bacteriol., 174:7716-7728), alkaline phosphatase, a tryptophan (trp) promoter system(Goeddel, (1980) Nucleic Acids Res. 8: 4057 and EP 36,776) and hybridpromoters such as the tac promoter (deBoer et al., (1983) Proc. Natl.Acad. Sci. USA, 80: 21-25). However, other known bacterial promoters aresuitable. Their nucleotide sequences have been published, therebyenabling a skilled worker operably to ligate them to DNA encoding thepolypeptide of interest (Siebenlist et al, (1980) Cell, 20: 269) usinglinkers or adaptors to supply any required restriction sites. See also,e.g., Sambrook et al., supra; and Ausubel et al., supra.

Promoters for use in bacterial systems also generally contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA.

(v) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) or other strains, and successful transformants are selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Sanger et al., (1977) Proc.Natl. Acad. Sci. USA, 74: 5463-5467 or Messing et al., (1981) NucleicAcids Res., 9: 309, or by the method of Maxam et al., (1980) Methods inEnzymology, 65: 499. See also, e.g., Sambrook et al., supra; and Ausubelet al., supra.

The nucleic acid encoding the heparin binding protein of interest isinserted into the host cells. Typically, this is accomplished bytransforming the host cells with the above-described expression vectorsand culturing in conventional nutrient media modified as appropriate forinducing the various promoters.

Culturing the Host Cells

Suitable prokayotic cells for use to express the heparin bindingproteins of interest are well known in the art. Host cells that expressthe recombinant protein abundantly in the form of inclusion bodies or inthe perplasmic or intracellular space are typically used. Suitableprokaryotes include bacteria, e.g., eubacteria, such as Gram-negative orGram-positive organisms, for example, E. coli, Bacilli such as B.subtilis, Pseudomonas species such as P. aeruginosa, Salmonellatyphimurium, or Serratia marcescens. One example of an E. coli host isE. coli 294 (ATCC 31,446). Other strains such as E. coli B, E. coliX1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable.These examples are illustrative rather than limiting. Strain W3110 is atypical host because it is a common host strain for recombinant DNAproduct fermentations. In one aspect of the invention, the host cellshould secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strains 1A2, 27A7, 27B4, and 27C7 described in U.S. Pat. No. 5,410,026issued Apr. 25, 1995. For example, a strain for the production of VEGFis E. coli stain W3110 having the genotype tonAΔ ptr3 phoAΔE15Δ(argF-lac)169 degP41 ilvg designated 49B3. In another example, a strainfor the production of VEGF is the E. coli strain (62A7) having thegenotype ΔfhuA (ΔtonA) ptr3, lacI^(q), lacL8, ΔompT Δ(nmpC-fepE) ΔdegPilvG⁺. See also, e.g., table spanning pages 23-24 of WO2004/092393.

Prokaryotic cells used to produce the heparin binding protein ofinterest are grown in media known in the art and suitable for culture ofthe selected host cells, including the media generally described bySambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y.) (2001). Media thatare suitable for bacteria include, but are not limited to, AP5 medium,nutrient broth, Luria-Bertani (LB) broth, Neidhardt's minimal medium,and C.R.A.P. minimal or complete medium, plus necessary nutrientsupplements. In certain embodiments, the media also contains a selectionagent, chosen based on the construction of the expression vector, toselectively permit growth of prokaryotic cells containing the expressionvector. For example, ampicillin is added to media for growth of cellsexpressing ampicillin resistant gene. Any necessary supplements besidescarbon, nitrogen, and inorganic phosphate sources may also be includedat appropriate concentrations introduced alone or as a mixture withanother supplement or medium such as a complex nitrogen source.Optionally the culture medium may contain one or more reducing agentsselected from the group consisting of glutathione, cysteine, cystamine,thioglycollate, dithioerythritol, and dithiothreitol.

Examples of suitable media are given in U.S. Pat. Nos. 5,304,472 and5,342,763. C.R.A.P. phosphate-limiting media consists of 3.57 g(NH₄)₂(SO₄), 0.71 g Na citrate-2H₂O, 1.07 g KCl, 5.36 g Yeast Extract(certified), 5.36 g HycaseSF™-Sheffield, adjusted pH with KOH to 7.3, qsvolume adjusted to 872 ml with deionized H₂O and autoclaved; cooled to55° C. and supplemented with 110 ml 1 M MOPS pH 7.3, 11 ml 50% glucose,7 ml 1M MgSO₄). Carbenicillin may then be added to the induction cultureat a concentration of 50 μg/ml.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the temperature ranges from, e.g., about 20°C. to about 39° C., or from about 25° C. to about 37° C., or at about30° C.

Where the alkaline phosphatase promoter is employed, E. coli cells usedto produce the polypeptide of interest of this invention are cultured insuitable media in which the alkaline phosphatase promoter can bepartially or completely induced as described generally, e.g., inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y.) (2001). The culturingneed never take place in the absence of inorganic phosphate or atphosphate starvation levels. At first, the medium contains inorganicphosphate in an amount above the level of induction of protein synthesisand sufficient for the growth of the bacterium. As the cells grow andutilize phosphate, they decrease the level of phosphate in the medium,thereby causing induction of synthesis of the polypeptide.

If the promoter is an inducible promoter, for induction to occur,typically the cells are cultured until a certain optical density isachieved, e.g., a A₅₅₀ of about 200 using a high cell density process,at which point induction is initiated (e.g., by addition of an inducer,by depletion of a medium component, etc.), to induce expression of thegene encoding the polypeptide of interest.

Any necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art,introduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. The pH of the medium may be any pH fromabout 5-9, depending mainly on the host organism. For E. coli, the pHis, e.g., from about 6.8 to about 7.4, or about 7.0.

Formulations of Heparin Binding Proteins

The polypeptide recovered, e.g., using the methods described herein, maybe formulated in a pharmaceutically acceptable carrier and is used forvarious diagnostic, therapeutic, or other uses known for such molecules.For example, VEGF described herein can be used in immunoassays, such asenzyme immunoassays. Therapeutic uses for the heparin binding proteinsobtained using the methods described herein are also contemplated. Forexample, a growth factor or hormone, e.g., VEGF, can be used to enhancegrowth as desired. For example, VEGF can be used to promote woundhealing of, e.g., an acute wound (e.g., burn, surgical wound, normalwound, etc.) or a chronic wound (e.g., diabetic ulcer, pressure ulcer, adecubitus ulcer, a venous ulcer, etc.), to promote hair growth, topromote tissue growth and repair (e.g., bone, liver, etc.), etc.

Therapeutic formulations of heparin binding proteins are prepared forstorage by mixing a molecule, e.g., a polypeptide, having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 18thedition, Gennaro, A. Ed. (1995)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In certain embodiments, the formulations to be used for in vivoadministration are sterile. This is readily accomplished by filtrationthrough sterile filtration membranes. HBP can be stored in lyophilizedform or as an aqueous solution or gel form. The pH of the HBPpreparations can be, e.g., from about 5 to 8, although higher or lowerpH values may also be appropriate in certain instances. It will beunderstood that use of certain of the excipients, carriers, orstabilizers can result in the formation of salts of the HBP.

Typically for wound healing, HBP is formulated for site-specificdelivery. When applied topically, the HBP is suitably combined withother ingredients, such as carriers and/or adjuvants. There are nolimitations on the nature of such other ingredients, except that theymust be pharmaceutically acceptable and efficacious for their intendedadministration, and cannot significantly degrade the activity of theactive ingredients of the composition. Examples of suitable vehiclesinclude ointments, creams, gels, sprays, or suspensions, with or withoutpurified collagen. The compositions also may be impregnated into steriledressings, transdermal patches, plasters, and bandages, optionally inliquid or semi-liquid form.

For obtaining a gel formulation, the HBP formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as polyethylene glycol to forma gel of the proper viscosity to be applied topically. Thepolysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrins; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. In certain embodiments of theinvention, the gelling agent herein is one that is, e.g., inert tobiological systems, nontoxic, simple to prepare, and/or not too runny orviscous, and will not destabilize the HBP held within it.

In certain embodiments, the polysaccharide is an etherified cellulosederivative, in another embodiment one that is well defined, purified,and listed in USP, e.g., methylcellulose and the hydroxyalkyl cellulosederivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose,and hydroxypropyl methylcellulose. In one embodiment, methylcellulose isthe polysaccharide. If methylcellulose is employed in the gel, e.g., ittypically comprises about 2-5%, or about 3%, or about 4% or about 5%, ofthe gel, and the HBP is present in an amount of about 300-1000 mg per mlof gel.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400-600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

The active ingredients may also be entrapped in microcapsules, orsustained-release preparations. See, e.g., Remington's PharmaceuticalSciences 18th edition, Gennaro, A. Ed. (1995). See also Johnson et al.,Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993);Hora et al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; U.S. Pat. No.5,654,010; DE 3,218,121; Epstein et al., (1985) Proc. Natl. Acad. Sci.USA, 82: 3688-3692; Hwang et al., (1980) Proc. Natl. Acad. Sci. USA, 77:40304034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Recombinant Human VEGF Expressed in Escherichia coli

Recombinant human VEGF was expressed in Escherichia coli. Duringsynthesis, the protein was secreted into the periplasmic space andaccumulated as refractile bodies. Studies were therefore conducted toachieve extraction and refolding of the protein. These studies revealedat least 3 species of VEGF (FIG. 1) were isolated using standardrecovery techniques without the addition of a polyanionic agent. Studieswith native VEGF showed that heparin addition increased resistance tochaotrope- and thiol-induced denaturation (FIG. 2). In addition, heparinsignificantly increased the amount of properly refolded VEGF in smallscale refolding experiments. To adapt this result to a large-scaleprocess, conditions were discovered which allowed for refolding VEGF inthe presence of dextran sulfate, a molecule structurally analagous toheparin. Addition of dextran sulfate improved yields of properly foldedbiologically active VEGF 3-5-fold relative to controls.

Methods

Plasmid for VEGF₁₆₅ expression—The plasmid pVEGF171 was designed for theexpression of human VEGF₁₆₅ (see, e.g., Leung et al., (1989) Science,246:1306-1309) in the E. coli periplasm. Transcription of the VEGFcoding sequence was placed under tight control of the alkalinephosphatase (AP) promoter (see, e.g., Kikuchi et al., (1981) NucleicAcids Research, 9:5671-8), while sequences required for translationinitiation were provided by the trp Shine-Dalgarno region (see, e.g.,Yanofsky et al., (1981) Nucleic Acids Research, 9:6647-68). The VEGFcoding sequence was fused downstream of the bacterial heat-stableenterotoxin II (STII) signal sequence (see, e.g., Lee et al., (1983)Infect. Immun. 42:264-8; and, Picken et al., (1983) Infect. Immun.42:269-75) for subsequent secretion into the E. coli periplasm. Codonmodifications in the STII signal sequence provided for an adjustedtranslation level, which resulted in an optimal level of VEGFaccumulation in the periplasm (see, e.g., Simmons and Yansura, (1996)Nature Biotechnoloy, 14:629-34). The lambda to transcriptionalterminator (see, e.g., Scholtissek and Grosse, (1987) Nucleic AcidsResearch 15:3185) was located downstream of the VEGF translationaltermination codon. The replication origin, and both ampicillin andtetracycline resistance genes, were provided by the plasmid pBR322. See,e.g., Bolivar et al., (1977) Gene 2:95-113.

Cell Homogenization and Refractile body preparation—HarvestedEscherichia coli cells were frozen and stored at −70 C.°. Cells wereharvested by BTUX (centrifuge, Alfa laval) centrifugation and freezingusing BEPEX (freezer at large scale). Cells were suspended in 5 volumesof 50 mM HEPES/150 mM NaCl/5 mM EDTA pH 7.5 (5 L/kg pellet) andhomogenized in a model 15 M laboratory homogenizer Gaulin 15M (smallscale) or M3 (large scale) (Gaulin Corporation, Everett, Mass.). Thecell suspension was then diluted with an equal volume of the same bufferand refractile bodies were harvested by centrifugation in a BTPX 205(Alfa Laval Separation AB(Tumba, Sweden) continuous feed centrifuge.Intermediate scale centrifuge used SA1. Alternatively, cells can behomogenized and the pellet can be harvested directly without freezing inBEPEX and rehydrating.

Example II Extracting and Refolding of Recombinant Human VEGF Expressedin Escherichia coli-I Methods

Extraction and Refolding—The refractile pellet was suspended inextraction buffer containing 7 M Urea/50 mM HEPPS/pH 8 (finalconcentration) at 5 L of buffer/kg pellet. Solid dithiothreitol was thenadded at 3.7 g/kg pellet for a final concentration of 4 mM. See, e.g.,FIG. 9 for the effect of urea and DTT on extraction of VEGF. Thesuspension was thoroughly mixed for 1-2 h at 20° C. The pH may beadjusted with 50% sodium hydroxide (w/w) to pH 8.0. Refolding wasinitiated by addition of 19 volumes of refolding buffer per volume ofextraction buffer. The refolding buffer contained 50 mM HEPPS/1 M-2MUrea/2-5 mM cysteine/0.05%-0.2% TRITON™ X100/pH 8, final concentration.See, e.g., FIG. 10 for the effect of urea and DTT concentration presentduring refolding. Dextran sulfate, heparin or sodium sulfate was addedas indicated. Refold incubation was conducted at room temperature for4-24 hours. Optionally, the incubation can be conducted at roomtemperature for up to about 48 hours. The folding was monitored bySDS-PAGE and/or Heparin HPLC. The product was clarified by depthfiltration with a Cuno 90SP filter followed by 0.45 μm filtration.

Heparin-binding HPLC Assay—The quality and quantity of properly refoldedVEGF was determined using a column containing immobilized heparin. Thecolumn POROS HE2/M (4.6×100 mm, HE2/M by PerSeptive BioResearchProducts, Cambridge, Mass.) was equilibrated in 10 mM sodium phosphate,pH 7 containing 0.15 M sodium chloride. At a flow rate of 1 mL/min or 2ml/min, the columns were eluted using a linear gradient from 0.15 M to 2M sodium chloride in equilibration buffer over 10 min. In some assays,elution was done in 16 min. The eluant was monitored at 280 nm.Typically, the majority of protein was eluted in the void volume and 3classes of VEGF could be identified. The highest affinity,latest-eluting species was identified as correctly folded VEGF and wassubsequently identified as “Peak 3 VEGF”.

Results

Heparin protects VEGF against cysteine-mediated denaturation—Addition of10 mM cysteine to native VEGF resulted in a large decrease in theproperly-folded molecule (FIG. 2). This denaturation was prevented bythe addition of 2 different forms of heparin at concentrations as low as20 mM. TABLE Ia The Effect of Heparin and Dextran sulfate on VEGFRefolding Concentration (μg/ml) Addition 0 10 55 100 200 400 % Increaseor Fold Increase None 5.3* — — Low (3 kd) MW heparin 12.2 14.2 14.8 14.1179% 2.8 High MW (6 kD) heparin 15.3 16.6 13.9 15.3 213% 3.1 Dextransulfate (10 Kd) 15.9 15.4 13.6 7.4 8.3 191% 2.9Values in the table are the amount of Peak 3 VEGF formed (in mg) per gof retractile pellet. Concentration of each addition is as indicated.*Average control (5.6 + 5.0 = 5.3)

TABLE Ib The Effect of Sodium Sulfate on VEGF Refolding Concentration(μg/ml) Addition 0 50 98 195 293 455 % Increase or Fold Increase None5.3 — — sodium sulfate 6.9 9.1 10.4 10.9 10.4 106% 2.1Values in the table are the amount of Peak 3 VEGF formed (in mg) per gof refractile pellet. Concentration of sodium sulfate is as indicated

TABLE II The Effect of Heparins and Dextran sulfates on VEGF RefoldingConcentration (μg/ml) Addition 0 2.5 12.5 50 100 % Increase or FoldIncrease None 2.2 — — dextran sulfate (5 Kd) 10.1 13.7 13.4 11.2 523%6.2 dextran sulfate (8 Kd) 9.9 17.2 14.0 12.9 682% 7.8 dextran sulfate(10 Kd) 13.8 19.2 14.6 10.1 773% 8.7 Low MW (3 Kd) Heparin 10.4 16.914.7 668% 7.7 High MW (6 Kd) Heparin 14.1 18.8 20.2 818% 9.2Values in the table are the amount of Peak 3 VEGF formed (in mg) per gof refractile pellet.Summary

Heparin and dextran sulfate increase refolding yields—Due to theprotective properties against denaturation described above, the effectof several different forms of sulfated polymers on refolding VEGF wasinvestigated. As seen in TABLE Ia (and in FIG. 5), both low and highmolecular weight forms of heparin increased the yield of refolded VEGFapproximately 3-fold. As seen in TABLE Ib (and in FIG. 6), sodiumsulfate increased the yield of refolded VEGF by approximately 2-fold.The 10 Kd form of dextran sulfate was also effective at increasingrefold yields; however, the higher concentration range investigated leadto substrate inhibition. Further investigation demonstrated that 5 Kd, 8Kd and 10 Kd forms of dextran sulfate all significantly increased theyield of VEGF on refolding (TABLE II). See FIG. 7. See also FIG. 8.

Example III Effect of Different Buffers and TRITON™ X-100 on theRecovery of VEGF Results

Buffer VEGF (mg/g pellet) HEPES, pH 8 13.3 HEPES, pH 8 with TRITON ™14.3 HEPPS, pH 8 16.6 TrisHCl, pH 8 12.8 HEPES, pH 7.2 9.1 HEPPS, pH 7.210.7 HEPES, pH 8 10.3 HEPPS, pH 8 12.8 HEPES, pH 8 + TRITON ™ X-100 12.4HEPPS, pH 8 + TRITON ™ X-100 13.9Summary

The combined data of Example I, II and III demonstrate a significant(2-5 fold) improvement in yield by including either heparin sulfates ordextran sulfates when refolding VEGF, a heparin-binding growth factor aswell as the conditions of recovery. This method has been implementedsuccessfully at industrial scale. It is expected that that this methodis applicable in the refolding of other basic growth factors and otherproteins that bind heparin.

Example IV Extracting and Refolding of Recombinant Human VEGF Expressedin Escherichia coli-II Methods

Extraction and Refolding—The refractile pellet was suspended inextraction buffer where the final concentration was 7 M Urea, 2-30 mMDTT (e.g., 10 mM DTT), 50 mM HEPPS/pH 7-9 (e.g., pH 8) at 5 L ofbuffer/kg pellet. The suspension was thoroughly mixed for 1-2 h at roomtemperature. Refolding was initiated by addition of 19 volumes ofrefolding buffer per volume of extraction buffer. The refolding buffercontained as the final concentration 1 M or 1.3 M urea, 2-15 mM cysteine(e.g., 7.5 mM cysteine), 0.5 mM DTT, 0-0.75 M arginine (e.g., 100 mMarginine), 15 mg/L dextran sulfate, 50 mM HEPPS, 0.05% TRITON™ X100/pH8. See, e.g., FIG. 12 for the effect on refolding in the presence ofcharged amino acids, where the addition of histidine produced the sameeffect as without amino acid additives. Refold incubation was conductedat room temperature for 12-24 hours. Optionally, the incubation can beconducted at room temperature for up to about 48 hours. Optionally, airor oxygen can be added during the refolding process (0.3-1 cc/min/L).The folding was monitored by SDS-PAGE and/or Heparin HPLC. The productwas clarified by depth filtration with a Cuno 90SP filter followed by0.45 μm filtration.

The overall dilution of the extraction and refolding steps was 1:100.Increasing the overall dilution of the extraction and refolding steps,e.g., to 1:100 to 1:200, increased the total amount of active VEGFalthough the concentration is lower. See FIG. 13.

The efficiency of refolding can be determined by determining the amountof dimer/monomer, where monomers can be determined by a C18reverse-phase HPLC column and dimer formation can be determined byheparin column chromatography or SP-5PW cation exchange chromatographyassay.

Example V Large-Scale Refolding

In order to test the scalability of the optimized refolding conditions,studies were conducted to examine the kinetics of refolding at small(0.1 L), intermediate (1 L) and pilot plant (250 L to 400 L) scale. Asshown in FIG. 4, the kinetics of refolding at large scale wereindistinguishable from the smaller scales and the final titer ofrefolded VEGF was slightly increased. These data demonstrate thescalablility of refolding with dextran sulfate. The product was furtherclarified by depth filtration with a Cuno 90SP filter followed by 0.45μm filtration.

Example VI Purification I of rhVEGF after Refolding

MacroPrep Ceramic Hydroxyapatite Chromatography—After refolding,insoluble material in the pool was removed by depth filtration. Theclarified pool was then loaded on to a ceramic hydroxyapatite column(35D×31H=30L) (Bio Rad, Hercules, Calif.) equilibrated in 50 mMHEPPS/0.05% TRITON™ X100/pH 8. The non-binding protein was removed bywashing with equilibration buffer and the VEGF eluted using an isocraticstep of 50 mM HEPPS/0.05% TRITON™ X100/0.15 M sodium phosphate/pH 8. Theflow rate was 120 cm/hr. Pooling fractions were determined by HeparinHPLC analysis of fractions.

Butyl SEPHAROSE™ Fast Flow Chromatography—The pool of VEGF was loadedonto a column of Butyl SEPHAROSE™ Fast Flow (35 D×26H=25L) (GEHealthcare, Uppsala, Sweden) equilibrated in 50 mM HEPPS/0.05% TRITON™X100/0.15 M sodium phosphate/pH 8. The flow rate was 100 cm/hr. Thecolumn was washed with equilibration buffer and the VEGF collected inthe column effluent. Fractions were collected and protein containingfractions were pooled, by measuring A280 nm.

Macro Prep High S Chromatography—The Butyl SEPHAROSE™ pool was loadedonto a column of Macro Prep High S (30D×39H=27 L) (BioRad, Hercules,Calif.) that was equilibrated in 50 mM HEPES/pH 8. After washing theeffluent absorbance at 280 nm to baseline, the column was washed withtwo column volumes of 50 mM HEPES/0.25 M sodium chloride/pH 8. The VEGFwas eluted using a linear, 8-column-volume gradient from 0.25-0.75 Msodium chloride in 50 mM HEPES/pH 8. The flow rate was 75-200 cm/hr.Fractions were collected and those which contained properly-folded VEGF,as determined by a heparin-binding assay, e.g., Heparin HPLC, werepooled.

Phenyl 5PW TSK Chromatography—The Macro Prep High S pool was conditionedwith an equal volume of 50 mM HEPES/0.8 M sodium citrate/pH 7.5. Theconditioned pool was then loaded on to a column of Phenyl 5PW TSK(18D×43H=11L) (Tosohaas, Montgomeryville, Pa.) that was equilibratedwith 50 mM HEPES/0.4 M sodium citrate/pH 7.5. After washing non-bindingprotein through the column with equilibration buffer, the VEGF waseluted from the column using a 10-column-volume gradient from 0.4-0 Msodium citrate in 50 mM HEPES, pH 7.5. Fractions were assayed bySDS-polyacrylamide gel electrophoresis and those containing VEGF ofsufficient purity were pooled.

Ultrafiltration/Diafiltration—The pooled VEGF was ultrafiltered on a 5kD regenerated cellulose membrane (G30619); Unit Pellicon; Feed Rate17.1 L/min. The membrane was conditioned with polysorbate 20. The pooledVEGF was ultrafiltered at a concentration of 6 g/L (UF1). The sample wasdiafiltrated with 7-14 DV (Diavolume) with 5 mM Sodium Succinate/275 mMTrehalose/pH 5.0. The final formulation was 5 mM Sodium Succinate/275 mMTrehalose/0.01% polysorbate 20/pH5.0, at a concentration of 5 mg/ml.

Example VII Purification II of rhVEGF after Refolding

Cation Exchange Liquid Chromatography—After refolding, insolublematerial in the pool can be removed by depth filtration. The refold poolis conditioned to pH 5.0-7.5 and about 2 to 6.5 mS/cm. In oneembodiment, the pool is conditioned to pH 6.5 and 5 mS/cm. The refoldpool can be then loaded on to a sulfopropyl extreme load column (SPXL)and eluted using a gradient of increasing salt concentration. Poolingfractions can be determined by Heparin HPLC analysis of fractions.

Hydrophobic Interaction Column (HIC):—The SPXL elution pool of VEGF canbe conditioned to 50 mS/cm for loading onto a Phenyl TSK chromatographycolumn (Tosohaas, Montgomeryville, Pa.). Fractions are collected andprotein containing fractions are pooled.

IEX or mixed mode:—The Phenyl TSK pool can be loaded onto a column ofion exchange chromatography (IEX) or mixed-mode chromatography.Fractions are collected and those which contained properly-folded VEGF,as determined by assays described herein are pooled.

Ultrafiltration/Diafiltration—The pooled VEGF can be ultrafiltered on a5 kD regenerated cellulose membrane (G30619); Unit Pellicon; Feed Rate17.1 L/min. For example, the membrane is conditioned with polysorbate20. The pooled VEGF is ultrafiltered at a concentration of 6 g/L (UF1).The sample is diafiltrated with 7-14 DV (Diavolume) with 5 mM SodiumSuccinate/275 mM TrehaloseipH 5.0.

In methods and processes described herein, final purity and/or activitycan be assessed by peptide mapping, disulfide mapping, SDS-PAGE (bothreduced and non-reduced), circular dichroism, limulus amobocyte lysate(LAL), heparin chromatography, heparin HPLC (e.g., Heparin HPLC can beused to determine VEGF dimer concentration), reverse phase (rp) HPLCchromatography (e.g., rpHPLC can be used to determine VEGF monomerconcentration), heparin binding, receptor binding (for example for VEGFe.g., KDR receptor binding-Bioanalytic R&D, and/or Flt1 receptorbinding), SEC Analysis, cell assays, HUVEC potency assays, ELISAs withVEGF antibodies, mass spec analysis, etc.

It is understood that the deposits, examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications,citations, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

1. A process for recovering a heparin binding protein from a prokaryoticcell culture, the process comprising the steps of (a) isolating saidheparin binding protein from the periplasm of said prokaryotic cellculture; (b) denaturing said isolated heparin binding protein in a firstbuffered solution comprising a chaotropic agent and a reducing agent;(c) incubating said denatured heparin binding protein in a secondbuffered solution comprising a chaotropic agent and a sulfatedpolyanionic agent for such a time and under such conditions thatrefolding of the heparin binding protein occurs; and (d) recovering saidrefolded heparin binding protein, wherein there is about a 2 to 5-foldincrease in refolded heparin binding protein recovered compared toincubating with no sulfated polyanionic agent.
 2. The process of claim1, wherein the heparin binding protein is a heparin binding growthfactor.
 3. The process of claim 2, wherein the heparin binding growthfactor is vascular endothelial growth factor (VEGF).
 4. The process ofclaim 3, wherein the VEGF is VEGF₁₆₅.
 5. The process of claim 1, whereinthe sulfated polyanionic agent is between about 3,000 daltons and 10,000daltons.
 6. The process of claim 3, wherein said second bufferedsolution comprises a dextran sulfate.
 7. The process of claim 3, whereinsaid second buffered solution comprises sodium sulfate.
 8. The processof claim 3, wherein said second buffered solution comprises heparin. 9.The process of claim 6, wherein the dextran sulfate is between about8,000 and 10,000 daltons.
 10. The process of claim 3, wherein said firstand second buffered solutions comprise HEPPS pH 8.0.
 11. The process ofclaim 1, wherein said second buffered solution further comprises areducing agent.
 12. The process of claim 11, wherein the reducing agentof the second buffered solution comprises a combination of cysteine andDTT.
 13. The process of claim 1, wherein said second buffered solutionfurther comprises a nonionic detergent.
 14. The process of claim 1,wherein said second buffered solution further comprises arginine and/orlysine.
 15. The process of claim 1, wherein said recovery step (d)comprises sequentially contacting said refolded heparin binding proteinto a hydroxyapatite chromatographic support, a first hydrophobicinteraction chromatographic support; a cationic chromatographic support,and a second hydrophobic interaction chromatographic support, andselectively eluting the heparin binding protein from each support. 16.The process of claim 15, wherein said first and second hydrophobicinteraction chromatographic support is selected from the groupconsisting of butyl-, propyl-, octyl- and aryl-agarose resins.
 17. Theprocess of claim 15, wherein said first hydrophobic interactionchromatographic support is a butyl-agarose support and said secondhydrophobic interaction chromatographic support is a phenyl-agarosesupport resin.
 18. The process of claim 1, wherein said recovery step(d) comprises sequentially contacting said refolded heparin bindingprotein to a cation exchange support; a hydrophobic interactionchromatographic support, and an ion exchange chromatographic support,and selectively eluting the heparin binding protein from each support.19. A method for recovering a heparin binding protein from a prokaryoticcell culture, the method comprising the steps of (a) isolating saidheparin binding protein from the periplasm of said prokaryotic cellculture; (b) denaturing said isolated heparin binding protein in a firstbuffered solution comprising a chaotropic agent and a reducing agent;(c) incubating said denatured heparin binding protein in a secondbuffered solution comprising a chaotropic agent and a sulfatedpolyanionic agent for such a time and under such conditions thatrefolding of the heparin binding protein occurs, wherein there is abouta 2 to 5-fold increase in refolded heparin binding protein recoveredcompared to incubating with no sulfated polyanionic agent; and (d)sequentially contacting said refolded heparin binding protein with ahydroxyapatite chromatographic support, a first hydrophobic interactionchromatographic support, a cationic chromatographic support, and asecond hydrophobic interaction chromatographic support, and selectivelyeluting the heparin binding protein from each support.
 20. A method forpurifying a heparin binding protein, the method comprising the steps ofsequentially contacting a refolded heparin binding protein with ahydroxyapatite chromatographic support, a first hydrophobic interactionchromatographic support, a cationic chromatographic support, and asecond hydrophobic interaction chromatographic support, and selectivelyeluting the heparin binding protein from each support.
 21. A method forrecovering a heparin binding protein from a prokaryotic cell culture,the method comprising the steps of (a) isolating said heparin bindingprotein from the periplasm of said prokaryotic cell culture; (b)denaturing said isolated heparin binding protein in a first bufferedsolution comprising a chaotropic agent and a reducing agent; (c)incubating said denatured heparin binding protein in a second bufferedsolution comprising a chaotropic agent and a sulfated polyanionic agentfor such a time and under such is conditions that refolding of theheparin binding protein occurs, wherein there is about a 2 to 3-foldincrease in refolded heparin binding protein recovered compared toincubating with no sulfated polyanionic agent; and, (d) sequentiallycontacting said refolded heparin binding protein with a cation exchangesupport; a hydrophobic interaction chromatographic support, and an ionexchange chromatographic support, and selectively eluting the heparinbinding protein from each support.
 22. The method of claim 19 or 21,wherein the polyanionic agent is between about 3,000 daltons and 10,000daltons.
 23. A method for purifying a heparin binding protein, themethod comprising the steps of sequentially contacting a refoldedheparin binding protein with a cation exchange support; a hydrophobicinteraction chromatographic support, and an ion exchange chromatographicsupport, and selectively eluting the heparin binding protein from eachsupport.